key: cord-0902979-3xtb8gye
authors: Rabets, A.; Bila, G.; Grytsko, R.; Samborsky, M.; Rebets, Y.; Vari, S.; Pagneux, Q.; Barras, A.; Boukherroub, R.; Szunerits, S.; Bilyy, R.
title: Development of antibodies to pan-coronavirus spike peptides in convalescent COVID-19 patients
date: 2020-08-22
journal: nan
DOI: 10.1101/2020.08.20.20178566
sha: a2ed9c9c7caeef02e4e30329ad935b592b324184
doc_id: 902979
cord_uid: 3xtb8gye

Coronaviruses are sharing several protein regions notable the spike protein (S) on their enveloped membrane surface, with the S1 subunit recognizing and binding to the cellular receptor, while the S2 subunit mediates viral and cellular membrane fusion. This similarity opens the question whether infection with one coronavirus will confer resistance to other coronaviruses? Investigating patient serum samples after SARS-CoV-2 infection in cross-reactivity studies of immunogenic peptides from Middle East respiratory syndrome coronavirus (MERS-CoV), we were able to detect the production of antibodies also recognizing MERS virus antigens. The cross-reactive peptide comes from the heptad repeat 2 (HR2) domain of the MERS virus spike protein. Indeed, the peptide of the HR2 domain of the MERS spike protein, previously proven to induce antibodies against MERS-CoV is sharing 74% homology with the corresponding sequence of SARS-CoV-19 virus. Sera samples of 47 convalescent SARS-CoV-2 patients, validated by RT-PCR-negative testes 30 days post-infection, and samples of 40 sera of control patients (not infected with SARS-CoV-2 previously) were used to establish eventual cross-bind reactivity with the MERS peptide antigen. Significantly stronger binding (p<0.0001) was observed for IgG antibodies in convalescent SARS-CoV-2 patients compared to the control group. If used as an antigen, the peptide of the HR2 domain of the MERS spike protein allows discrimination between post-Covid populations from non-infected ones by the presence of antibodies in blood samples. This suggests that polyclonal antibodies established during SARS-CoV-2 infection has the ability to recognize and probably decrease infectiveness of MERS-CoV infections as well as other coronaviruses. The high homology of the spike protein domain suggests in addition that the opposite effect can also be true: coronaviral infections producing cross-reactive antibodies affective against SARS-CoV-19. The collected data prove in addition that despite the core HR2 region being hidden in the native viral conformation, its exposure during cell entry makes it highly immunogenic. Since inhibitory peptides to this region were previously described, this opens new possibilities in fighting coronaviral infections.

Coronaviruses such as the Middle East respiratory syndrome coronavirus ( The spike surface glycoprotein (S) plays a key role in mediating virus attachment and fusion and are indeed present in all human infecting coronaviruses. They can be cleaved by host proteases into an N-terminal S1 subunit and a membrane-bound C-terminal S2 region. In order to engage a host receptor, the receptor-binding domain (RBD) of the S1 subunit undergoes conformational movements, which transiently hide or expose the determinants of receptor binding [3, 4] . The Heptad Repeat 1 (HR1) region in S2 subunits forms a homotrimeric structure, exposing 3 highly conserved hydrophobic grooves on the surface resulting in binding of 3 Heptad Repeat 2 (HR2) regions and formation of six-helix bundle structure (6-HB). 6-HB is responsible for close approximation of viral and host membranes and their subsequent merging. Binding of HR1 and HR2 domains results in the six-helix bundle needed for merging with host cell membrane. Thus parts of the HR2 domain specifically binding HR1 can be considered as ideal coronaviral inhibitor strategy preventing cellular entry [5] . Further optimization of peptide sequence resulted in pan-coronaviral inhibitors, like EK1 [3] able to inhibit SARS-CoV-19 pseudovirus infection [6] .

The structure of HR2 is poorly resolved during crystallographic assessment due to high level of flexibility [7] . Unlike highly mutable receptor-binding domain, the HR1 and HR2 domains are highly conservative between coronaviruses, so form a perfect target for viral neutralization and generation of immunity that latter can be used for viral testing. Since HR2 and HR1 domains are merged and surface exposed after S protein cleavage we expected them to be highly immunogenic.

This similarity can result in the development of cross-reactive antibodies and protection against other coronaviruses, in case of being infected by another virus species. In this work we would like to establish if SARS-CoV-2 results in production of antibodies, that are also recognizing MERS virus antigens.

The complete crystal structure of the HR2 domain of SARS-CoV-2 remains currently unavailable due to it conformation changes and problems in stabilization [4] (Figure 1a) . Using SEQATOMS algorithm [8] the most complete structure of the corresponding region containing defined atomic coordinates was identified to be the one proposed by Walls et al [9] , namely a model for HR1 HR2 rearrangements and unfolding accompanying viral entry into host cells for other coronaviruses. This structure clearly demonstrates exposure of the HR2 domains upon cellular binding in trimeric (Figure 1b) and monomeric form (Figure 1c) . To evaluate the cross reactivity we selected the HR2-specific peptide of the spike protein of MERS-CoV reported to possessed high immunogenic potential [10] [11] [12] . Indeed, ongoing studies showe that it can be also successfully used to raise MERS-recognizing antibodies in the presence of neutrophil extracellular traps(NET) forming nanoadjuvants [13] . The selected HR2 peptide of the spike protein of MERS virus (depicted yellow, Figure 1d using PDB deposited crystal structure of 4NJL_A, [14] ) shares significant similarities in 3D structure between (the only) known crystal structure of unfolded HR2 domain ( Figure 1c ) and with pan-coronaviral inhibitor peptide EK1, using crystal structure 5ZVK_a [3] (Figure 1e) . Protein BLAST analysis reveales 46% identity and 76% similarity in aminoacid sequence of the MERS peptide with the corresponding peptide of the SARS-CoV-19 spike protein (sequence ID QKJ68605.1) (Figure 1f ). All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 22, 2020. .

(a) Image of Spike protein (S1) of SARS-CoV-2. (b) S1 upon host cell interaction: conformational changes in trimer are occurring, exposing previously hidden HR2 domain regions [4] , (c)monomeric part of (b), exposing domain with structural similarity (yellowidentical, orangesimilar amino acids) towards corresponding MERS peptide, depicted on (d) and pan. (f). Protein BLAST All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 22, 2020. . analysis of used MERS protein is showing similarity towards the sequence in the genome of SARS-CoV-19. Sequence ID: QKJ68605.1. Crystal structure of HR2 domain for SARS-CoV-19 is currently not available, thus it is represented as rectangle in (a) based on last connected coordinates available in 6VSB structure.

Having established the structural similarity between the S1 MERS peptide with the genome of SARS-CoV-2, sera of convalescent SARS-CoV-2 infected patient, who have never suffered from MERS-CoV infection before, have been collected and tested for the presence of antibodies.

The S1

MERS-CoV specific peptide, NH2-CCTTTTTTSLTQINTTLLDLEYEMKKLEEVVKKLEESYIDLKEL-COOH, which we previously successfully used to raise anti-MERS antibodies while testing novel NET-stimulating adjuvants [13] , was immobilized on ELISA plates and incubated with sera samples. As can be seen from Figure S1 ).

Using this S1 MERS-CoV specific peptide, discrimination of persons that have suffered from SARS-CoV-2 infection and those who were not in contact with the virus resulted in a predictive value (area under ROC curve) equal to 0.823 (Figure 2b) , with a specificity and sensitivity of ~60% (95% confidence). SARS-CoV-2 infections results in the generation of antibodies with significantly strong cross-reactive towards a MERS specific peptide with 76% homology. Highly conservative region of the exposed domain suggest that the opposite can be truecoronaviral disease can result in some antibodies able to recognize SARS-CoV-19 epitops circulating in the blood.

To determine whether the strong binding with S1 peptide is correlated with higher amount of anti-SARS-CoV-19 antibodies we used recombinant RBD protein immobilization on ELISA plates to incubate with sera samples to evaluate the amount of formed IgG type antibodies. As can be seed from Figure 2c , the sera samples having shown stronger binding of IgG antibodies with anti-HR2 MERS spike protein also contained higher IgG reactivity towards anti-RBD spike protein of SARS-CoV-19. The Pearsons correlation between the two parameters was 0.5492, p<0.0001. This suggests stronger humoral responses towards one of the virus will be associated with the intensity of the immune response towards other coronaviruses.

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perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted August 22, 2020. . 

Cross-reactivity between coronaviruses has become a critical question, since it brings new promises against COVID-19 protection. On the other hand, this cross-reactivity can be negative, since available cross-reactivity towards coronaviruses will make coronaviruses not the best choice for vectors in vaccines, especially taking into account recent data on broad immune cross-reactivity [15] . Indeed, it was reported that epitope pools detect CD4+ and CD8+ T cells in 100% and 70% of convalescent COVID patients respectively, recognizing S and M proteins, with at least eight SARS-CoV-2 ORFs targeted. T cell reactivity to SARS-CoV-2 epitopes is also detected in non-exposed individuals [6] . In SARS-CoV-2 patients S-reactive CD4+ T cells equally target N-terminal and C-terminal parts of the spike protein, whereas in healthy donors S-reactive CD4+ T cells react almost exclusively to the C-terminal part. This part is characterized by a higher homology to spike glycoprotein of human endemic "common cold" coronaviruses, and contains the S2 subunit of S with the cytoplasmic peptide (CP), the fusion peptide (FP), and the transmembrane domain (TM) but not the receptor-binding domain (RBD). S-reactive CD4+ T cells from SARS-CoV-2 patients are further distinct to those from healthy donors as they co-expressed higher levels of CD38 and HLA-DR, indicating their recent in vivo activation [7] . Potential preexisting cross-reactive T cell immunity to SARS-CoV-2 has broad implications, as it could explain aspects of differential SARS-CoV-2 clinical perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted August 22, 2020. . https://doi.org/10.1101/2020.08.20.20178566 doi: medRxiv preprint outcomes, influence epidemiological models of herd immunity, or affect the performance of SARS-CoV-2 candidate vaccines. Pre-existing memory CD4+ T cells that are cross-reactive with comparable affinity to SARS-CoV-2 and the common cold coronaviruses HCoV-OC43, HCoV-229E, HCoV-NL63, or HCoV-HKU1. Thus, variegated T cell memory to coronaviruses that cause the common cold may underline at least some of the extensive heterogeneity observed in COVID-19 disease [8] .

Based on these and our data one can assume that the C-terminal part of the spike protein is immunogenic due to exposure upon merging with host cells. Its conservative nature provides the background for the development of cross-coronaviral immune responses both cellular, and demonstrated here, by a IgG type humoral immune response. perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted August 22, 2020. . https://doi.org/10.1101/2020.08.20.20178566 doi: medRxiv preprint Goat anti-human IgG (H+L)-horseradish peroxidase (HRP) (109-035-003, Jackson ImmunoResearch) was diluted in washing buffer (1:25000), added to the plates and incubated at room temperature for 1 h. After the corresponding washings, the assay was developed with 3,3',5,5'-tetramethybezidine (TMB) containing excess of H2O2 as a substrate (50 µL per well).

The reaction was stopped with 50 µl/well of sulfuric acid (1 M). The absorbance was read at 450 nm/600nm using a Perkin Elmer BioAssay reader HST700 (Waltham, USA).

The protein homology searches were done using blast (NCBI) and PDB databases. In order to include the regions with resolved structures in our searches we had used SEQATOMS (http://www.bioinformatics.nl/tools/seqatoms/) [8] . Protein structures were visualized using PyMOL (https://pymol.org/). Multiple sequence alignments were done using CLUTALW [16] Data analysis. ELISA testing was performed in duplicate using 2 technical replicates for each analysis (coefficient of variation [CV] always <3%). The data were normalized between plates using positive controls and corrected for background signal of secondary antibodies, then the mean values were calculated and are shown on the graphs. For comparisons between two groups, the Mann-Whitney U-test for numerical variables was used. A receiver operating characteristic (ROC) curve was generated. The area under the ROC (AUROC) was calculated to estimate the specificity, sensitivity and usefulness of the binding assays. All analyses were perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted August 22, 2020. . https://doi.org/10.1101/2020.08.20.20178566 doi: medRxiv preprint Figure S1 . Antibody classes binding peptide of HR2 spike protein of MERS virus in convalescent COVID-19 sera, from left to right: binding of IgM, IgA and IgE immunoglobulins.

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preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted August 22, 2020. . https://doi.org/10.1101/2020.08.20.20178566 doi: medRxiv preprint

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